The overall objective of our research is to delineate the biochemical and genetic mechanisms by which the activity of asparagine synthetase is regulated in mammalian cells. Our previous work has demonstrated that in CHO cells enzyme activity is elevated in response to asparagine deprivation or to restricted aminoacylation of any of several tRNAs. In addition, we have isolated mutants resistant to Beta-aspartylhydroxamate which have constitutively depressed levels of asparagine synthetase. A monospecific antibody prepared against beef pancreas asparagine synthetase crossreacts with hamster enzyme and the enzyme from other mammalian species. Using this antibody we have shown that the increased asparagine synthetase activity in physiologically and genetically depressed cells is due to an increased rate of synthesis of a structurally normal enzyme. We now propose to quantitate the levels of asparagine synthetase mRNA in wildtype and physiologically or genetically depressed cells to determine the basis for this increased synthetic rate - steady state concentration of mRNA or translational control. Translational control could be studied further in a recently developed cellfree translation system prepared from CHO cells. Asparagine synthetase specific mRNA will be purified and cDNA prepared and used to study the mechanisms responsible for increased accumulation of mRNA by employing nucleic acid hybridization techniques. We also plan to isolate and characterize genetically and biochemically additional mutants affecting the expression of the asparagine synthetase structural gene in hamster cells. The biochemical techniques developed in the studies of CHO cells will be applied to an analysis of cultured leukemic and lymphoma cells which have an absolute requirement for exogenous asparagine and to prototrophic revertants which arise from these cells. The goal of these latter studies is to derive enough information about the regulation of asparagine biosynthesis in normal and malignant tissues to provide additional therapeutic approaches to be used in conjunction with asparagie therapy.